Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome. Most cases (approximately 70%) identified and characterized to date are associated with dominant germline mutations in the tumor suppressor gene TP53 (p53). Another tumor suppressor gene, CHEK2, was recently identified as a second minor predisposing locus. Studying a series of non-p53 LFS kindreds, we have shown that there is additional genetic heterogeneity in LFS kindreds with inherited predisposition at a locus other than p53 or CHEK2. Using a genome-wide scan for linkage with complementing parametric and non-parametric analysis methods, we have identified linkage to a separate, previously not implicated genomic region. In addition to a major predisposing locus, there is evidence for significant heterogeneity in risk within and between kindreds, in both p53 and non-p53 LFS kindreds. These data implicate additional risk modifiers in the genesis of LFS and its variants, including another major gene(s) as well as modifier genes. We hypothesize that the inherited susceptibility to childhood and associated cancers in non-p53, similar to p53, LFS kindreds is the result of a highly penetrant, dominantly acting gene. In keeping with a multi-step carcinogenesis model, however, germline mutations are not sufficient, and other modifier genes and factors, including epigenetic alterations, appear to be necessary in both p53 and non-p53 LFS kindreds. To test these hypotheses, we propose the following three specific aims that take advantage of the unique and large resources assembled as part of this program project: (1) to identify and characterize the gene for the newly mapped non-p53 LFS susceptibility locus; (2) to identify p53 and non-p53 LFS modifier genes; and (3) to evaluate the contribution of promoter hypermethylation and transcriptional inactivation of known cancer genes subject to epigenetic silencing to the LFS phenotype. Identification of the major non-p53 predisposing gene and its underlying mutations should provide insight into other genetic events that predispose to the genesis of diverse tumor types associated with LFS and its variants. Our integrated genomics approach that combines genomic and transcriptomic with epigenomic profiling will yield a better understanding of the complex molecular genetic and epigenetic events underlying the multi-step carcinogenesis in LFS and its variants and will provide valuable functional clues about potential candidate cancer and modifier genes, complex cellular candidate pathways, and the overall pathophysiology. As a childhood cancer, LFS is a unique model to study the underlying genetic events associated with a complex cancer syndrome, presumably because fewer such alterations are needed to give rise to the associated cancer. Similar to p53, other LFS predisposition and/or modifier genes may be functionally similarly important in other solid tumor types lacking a clear predisposition and inheritance pattern.